With the advent of the polymerase chain reaction, microbiologists were quick to realise that this new diagnostic modality had the potential to make microbiological diagnosis easier, cheaper and faster. Instead of conventional laboratory methods that rely on phenotypic expression of antigens or biochemical products, molecular methods provided a rapid identification of a number of infectious agents. Molecular methods have become increasingly incorporated into the clinical microbiology laboratory, particularly for the detection and characterisation of virus infections and for the diagnosis of diseases due to fastidious bacteria. The advantages were obvious, there was a rapid turn around time with a high sensitivity and specificity but the problems were of quality control and contamination.
Usually molecular methods involve the use of the PCR, either straightforward PCR's or the nested variety. In the future, it is hoped that the use of the microarray will further increase the utility of molecular methods.
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In this chapter, the use of molecular biology in the diagnosis of infectious diseases will be discussed. The chapter will also outline the problems and pitfalls. Lastly, the chapter will close with a brief word on the burning topic of today, biological warfare.
MOLECULAR BIOLOGY IN VIROLOGY -
CNS infections - Polymerase chain reaction has revolutionised the diagnosis of CNS infections. Previously, the diagnosis of CNS infections like Herpes Simplex virus (HSV) used to be cumbersome and insensitive. However, with the advent of the PCR, the diagnosis of HSV is relatively simple. HSV is the most common cause of acute sporadic focal encephalitis. The sensitivity of CSF PCR for the diagnosis of HSV is 96% and that the specificity is 99%. About the same percentages characterise molecular methods used to detect enteroviruses, Epstein-Barr virus and human cytomegalovirus in the CSF, whereas sensitivity and specificity rates decrease for the VZV, HHV6, HIV and rabies viruses. JCV is a slow-growing virus (up to five weeks), so a diagnostic test different from culturing is needed. Previously, the virus was searched by means of a nested PCR which had 92% sensitivity and 100% specificity. However, after the introduction of HAART therapy, the PCR technique showed a strong decrease in the sensitivity, because of the lower amount of virus in the CSF, due to the restoration of the immune system. However, with the help of the real time PCR, sensitivity and specificity has been restored to the previous levels.
HPV infection - Early diagnosis of papillomavirus infection is a key issue for the prevention of HPV-related cancers. In some developing countries, HPV related cancers represent the most prevalent neoplastic pathology. Screening programs have been, and are, mainly based on the examination of cytologic smears from the cervical canal stained by Papanicolau staining technique (Pap-test).
Since antibody response is unreliably detected and does not necessarily correlate with current viral presence, the only diagnostic virologic assays for HPV infection are based on HPV DNA detection and typing. Different PCR assays, mostly using general primers for the conserved L1 or E1 regions, have been developed. In some cases, nested protocols using combinations of the mentioned set of primers can be used. Typing is achieved by sequencing, restriction endonuclease digestion or probe hybridization, all performed on the amplified product. In spite of these advances, none of these methods can be considered a golden standard. Microarray based assays are currently being developed to improve specificity and type range.
Unlike cytologic screening, a swab drawn from anywhere in the vagina or from the vaginal
fornices, is adequate for obtaining a reliable result, since shed infected cells and virions, with their high load of viral DNA are spread throughout the genital tract.
Hepatitis viruses - Nucleic acid detection techniques are more sensitive than immunoassays for viral antigen to detect HAV in samples of different origins. Hepatitis A virus has been detected with techniques such as PCR - RFLP, SSCP, Southern blotting, sequencing, nucleic acid hybridization and reverse transcription-PCR (RT-PCR). Amplification of viral RNA by RT-PCR is currently the most sensitive and widely used method for detection of HAV RNA.
Diagnosis of hepatitis C is based on serological assays which detect HCV-specific antibodies (anti-HCV) and on molecular assays which detect HCV RNA. The molecular assays currently available are reverse transcriptase (RT)-PCR. Third-generation anti-HCV enzyme-linked immunosorbent assays (ELISAs) are highly sensitive as well as specific and represent the primary diagnostic assay. The recombinant immunoblot assay (RIBA) is a supplemental assay that can be used to confirm a positive ELISA, particularly in low-risk populations.
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HCV becomes positive by RT-PCR as early as 1-2 weeks after infection and 4-6 weeks before anti-HCV seroconversion. The determination of HCV RNA is, in principle, important for the selection of patients for antiviral therapy and for the assessment of its efficacy. In the case of a positive ELISA, RT PCR allows to discriminate between patients with chronic hepatitis C and those with resolved HCV infection that can remain anti-HCV positive for years or decades.
Discrimination of genotype 1 from genotypes 2 and 3 as well as quantitative determination of viremia levels has become important for the selection of the optimal treatment regimen. In general, however, genotyping and quantitative RT PCR tests should be used only in the context of a defined therapy protocol and not for the initial diagnosis of HCV infection.
Respiratory infections - Of the common viruses causing respiratory infections, the use of molecular methods in the diagnosis of adenovirus, influenza virus, parainfluenza virus, and respiratory syncytial virus (RSV) infections has not been clearly established. Although PCR techniques are available for the diagnosis of these viruses, other rapid conventional techniques are available: influenza virus and RSV can be detected in the clinical specimens by immunofluorescence and parainfluenza virus and adenovirus can be detected by immunofluorscence after incubation for 48 h in shell vial cultures. In these cases, nucleic acid amplification techniques have no added value in terms of sensitivity or rapidity.
Rhinoviruses and coronaviruses grow poorly in cell culture and rapid detection techniques like immunofluorscence and culture techniques are not available for these viruses. In such cases, molecular methods present a distinct advantage. The PCR is rapid and fairly sensitive.
Hantavirus pulmonary syndrome, is characterized by fever, myalgias, headache, and cough, followed rapidly by respiratory failure. Antibodies against heterologous hantavirus antigens were initially used to identify the causative agent, and then the hantavirus genome was detected by PCR in autopsy specimens. The virus cannot be cultured and so the PCR remains the only diagnostic possibility.
Besides these viruses, PCR based assays have been developed for the SARS and H5N1 strains of influenza as well.
Gastrointestinal disease - Viruses cause more infectious diarrhoea worldwide than bacteria and other pathogens. The method of choice for microbiological diagnosis of rotavirus from stool samples is PCR. Norovirus, a calicivirus formerly known as Norwalk virus can be diagnosed by electron microscopy, enzyme immunoassay and PCR but PCR is the most sensitive and rapid method. PCR is also the most sensitive method for the diagnosis of astroviruses and enteric adenoviruses (serotypes 40 and41).
HIV - Although infection with the human immunodeficiency virus (HIV) is routinely diagnosed by serology, early HIV infection can be detected by HIV pro-viral DNA detection before HIV antibodies are confirmed by Western Blot serology. Vertical transmission of HIV infection is also detected in the infant using HIV pro-viral DNA detection. These methods are capable of reducing the potentially infectious window period. The PCR is also used to quantitate the viral load. Since the viral load can be quantitated using real time PCR, molecular techniques can thus be used to prognosticate in cases of HIV infection. Finally, HIV genotyping for the detection of drug resistance is the standard of care to guide antiretroviral therapy and complements viral load assessment.
MOLECULAR BIOLOGY IN BACTERIOLOGY -
Molecular identification should be considered in three scenarios, namely (a) for the identification of an organism already isolated in pure culture, (b) for the rapid identification of an organism in a diagnostic setting from clinical specimens or (c) for the identification of an organism from non-culturable specimens, e.g. culture negative endocarditis.
Most modern clinical microbiology diagnostic laboratories rely on a combination of colonial morphology, physiology and biochemical/serological markers, for their successful identification to the genus and species levels. The importance of identifying organisms correctly is because it is necessary to correctly type bacteria for epidemiological purposes and for infection control purposes.
The rapid identification of an organism already isolated in pure culture needs no elaboration. Molecular methods can rapidly isolate the nucleic acid from the cultured organism and type it accurately and rapidly. For rapid identification of organisms in a diagnostic setting from clinical specimens, molecular biological techniques play an important role. The culture report may take a long time to come and so in many cases, patients maybe managed empirically. In today's world, the importance of rapid diagnosis is also in the setting of a bioterrorist attack.
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In some cases, the organism is non culturable. In such situations, molecular biology has an important role to play. Take a situation where antibiotic therapy has been instituted and the organisms cannot be cultured. Even in such cases, the organisms DNA remains intact and this DNA can be exploited in molecular diagnostics.
By and large, molecular techniques in bacteriology, use DNA as a starting material. DNA has the advantage of being stable and bacteria contain DNA s their genetic material. Usually, molecular biology is required to answer one question; whether or not the target DNA is present. Quantitation has little meaning unless some specific problem like food contamination is being studied. Under the circumstances, DNA is adequate as a starting material.
At times, there is no indication regarding the identity of a bacterial organism, employment of amplification of DNA encoding ribosomal RNA genes in conjunction with DNA sequencing of the amplicon can be done. In bacteria, there are three genes which make up the rRNA functionality, i.e. 5S, 16S and 23S rRNA. The 16S rRNA gene has historically been most commonly employed for identification purposes due to it being highly conserved and having a moderate copy number depending on the genus. 16S rRNA genes are found in all bacteria and
accumulate mutations at a slow, constant rate over time, hence they may be used as "molecular clocks". Highly variable portions of the 16S rRNA sequence provide unique signatures to any bacterium and useful information about relationships between them.
More recently, employment of the 16S-23S rRNA intergenic spacer region has become popular due to its high copy number and more importantly its high sequence variability. The 23S rRNA subunit has also been used to identify bacterial species. In times to come, it is likely that bacteriological diagnosis will look beyond just the 16S rRNA sequence. Finally, there are situations where the 16S rRNA sequence may not provide an adequate discrimination between species. Under the circumstances, sequences of essential genes like the heat shock proteins maybe employed. This is particularly useful in discriminating between species like Burkholderia cenocepacia and B. multivorans.
The diagnosis of infections due to fastidious bacteria has benefited greatly from molecular detection. Many of these fastidious bacteria have public health implications such as Mycobacterium tuberculosis, Chlamydiatrachomatis, Neisseria gonorrheae and Bordetella pertussis. Non-culture-based molecular testing has the advantage of avoiding the delays of days to weeks for conventional culture to allow early recognition and treatment as a public health imperative.
Fastidious bacteria usually include Mycobacterium tuberculosis and sexually transmitted diseases. Collection of samples in cases of sexually transmitted diseases can be a source of intense embarrassment. Again, the chances of contamination are quite high and most of the organisms are highly fastidious and require special conditions for growth. Under the circumstances, molecular detection is useful since noninvasive specimens unsuitable for traditional culture, such as initial stream urine and self-collected vaginal swabs can be used. These are more convenient and acceptable increasing the compliance with testing. Molecular testing methods give a sensitivity and specificity which is equivalent to what is seen with standard culture methods. In remote areas, molecular methods have the advantage of being performed on dry swabs with little degradation of the DNA during transit compared to the difficulties of transporting samples in specialised transport medium to preserve viability. In addition, molecular methods can test for multiple genital pathogens such as C. trachomatis, N. gonorrhoeae, the Donovanosis agent and the genital mycoplasmata from the same swab.
Mycobacteriology has been aided by the introduction of molecular methods. However, it is important to note that molecular detection of M. tuberculosis is one of the few examples where conventional culture remains more sensitive. This is possibly due to the difficulty in releasing the DNA from the bacterial cells during the extraction process. Yet, molecular methods are important in detection of M tuberculosis since it allows confirmation of acid-fast bacilli seen on microscopy with up to 98% sensitivity in pulmonary tuberculosis within a day compared to two weeks or more by culture. Specimens that are smear-negative have a much lower chance of molecular confirmation. Molecular biology can also speciate the organisms grown on a culture plate in a day as compared to the four weeks it would normally take by standard methods.
Antibiotic resistance markers
Antibiotic resistance is the burning topic today. With bacterial becoming resistant to antibiotics faster than antibiotics can be discovered, the detection of antibiotic resistance is of utmost importance.
Applying rapid and reliable genotypic detection to bacteria with infection control implications such as methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE) is of great potential benefit. The discrimination of MRSA from other S. aureus is confirmed by the detection of the mecA gene responsible for this resistance. It is important to detect MRSA early, not only for epidemiological purposes but for the patients treatment. MRSA requires more aggressive and specific treatment. Similarly, the detection of VRE is more sensitive and rapid using DNA-based amplification techniques.
Extended spectrum Î²-lactamases (ESBL) are found in Escherichia coli and Klebsiella pneumoniae and are readily transmitted on plasmids and transposons. ESBL-containing bacteria can spread rapidly in health care facilities to cause wound infections, urinary tract infections and septicaemia. Molecular detection of these point mutations at the active site of the Î²-lactamase gene can confirm the ESBL and allows for epidemiological typing.
Multi-drug resistant tuberculosis (defined as the presence of both rifampicin and isoniazid resistance) is a serious problem in many parts of the world. Rather than employ traditional culture methods which deliver results after several days, detection of the rpoB and hsp65 gene targets can detect the resistance genes in a single day.
MOLECULAR BIOLOGY IN MYCOLOGY AND PARASITOLOGY
Although not as frequently applied to eukaryotic infections, in a number of clinical circumstances molecular testing can be helpful. PCR can be used to detect Pneumocystis jiroveci infection in HIV patients. The specificity of PCR is limited, however, as this organism is a ubiquitous commensal and can be detected by PCR in the absence of pneumonia. Aspergillus spp can be detected by PCR especially in neutropenic patients. Aspergillus maybe very difficult to culture early in the disease and so molecular testing can be of great benefit.
Parasitological diagnosis is aided by molecular methods since most parasites are not cultured in routine laboratory settings and therefore diagnosis relies mostly on the relatively less
sensitive microscopy or serology. Toxoplasma gondii can be detected by PCR from amniocentesis fluid to confirm foetal infection and from CSF to diagnose toxoplasma encephalitis. In cases of malaria, PCR can diagnose malaria even after chemoprophylaxis and/ or treatment has been given. It can also diagnose mixed infections which are difficult to diagnose on microscopy.
LIMITATIONS OF MOLECULAR METHODS
Despite significant advantages of molecular diagnostics it cannot yet replace conventional methods for a range of infectious diseases since many common tests performed in the clinical microbiology laboratory are rapid and inexpensive. Culture methods have advanced considerably and the modern automated culture systems allow for rapid identification and susceptibility testing. Bacterial culture can detect a large number of bacteria and speciation can be done rapidly. In contrast, the PCR can only detect the organism whose DNA is complementary to the primers used. Therefore to cover a similar breadth of possible organisms as culture, it would require the introduction of inexpensive and simple microarray technologies that are not yet available.
False Positive and False Negative Results
Contamination remains the bugbear of molecular testing methods. The problems of contamination and laboratory management have been dealt with elsewhere in this book, suffice to say that scrupulous attention to contamination needs to be given. To avoid false positive results due to laboratory contamination relatively large laboratory areas are required for physical separation of reagent preparation, specimen preparation and product detection areas together
with a high level of staff training and skill. Amplicon laboratory contamination can be reduced by ultraviolet light irradiation of reagents and chemical inactivation of surface contamination with sodium hypochlorite. Intersample contamination can be reduced by the use of disposable equipment and cotton filter tips, and using disposable personal protective equipment such as caps, gowns and gloves. Appropriate negative controls are to be included in every PCR run to detect any contamination.
Poor primer design can also lead to erroneously positive results. Primers may be poorly designed such that incidental amplification of microorganisms other than those sought occurs. Also primers are designed based on the known sequences available through international databases but designing a primer wrongly may result in non specific amplification. For further clarification on this subject, readers are requested to refer to primer design in the basics of PCR.
False negative results may also be a problem. It maybe difficult to extract DNA from organisms like Mycobacterial spp. Substances in some clinical specimens such as sputum and faeces can degrade the DNA and RNA and other specimens may contain substances such as polysaccharides, haem and therapeutic drugs that inhibit the PCR enzymes. It is therefore important to include inhibitor checks for each specimen to ensure a negative PCR reaction is not actually an inhibited reaction. Internal controls can check for both the presence of inhibitors and for a successful DNA extraction.
Lack of Uniformity in Molecular Testing
Molecular diagnosis is also complicated by the vast array of in-house PCR tests used in different laboratories. Many tests are not available because it is simply not commercially viable to manufacture the kits. Investigators then begin to develop their own in house tests which use different primers amplifying different genes and/ or different sequences within genes. The PCR format maybe different (standard PCR, multiplex or nested PCR). These variables leads to a considerable lack of uniformity in testing.
Differentiation between Infection and Disease
Since the presence of nucleic acid does not necessarily mean the presence of viable organisms a problem with interpretation of PCR results can emerge that does not occur with culture. For some infections such as invasive meningococcal disease the presence of meningococcal DNA from a sterile site has a very high positive predictive value. However, the detection of P. jiroveci in suspected PCP may have only a 50% positive predictive value in immunosuppressed patients since it may colonise as well as cause disease. In some cases, quantitative PCR maybe helpful because higher organism loads are more specific for infection. Also, RNA can be used as a template. Since RNA degrades easily, the presence of RNA would indicate pathogen viability and replication.
MOLECULAR BIOLOGY AND BIOTERRORISM
The earlier belief and various hypotheses that bioterrorism is not a serious threat has been proved wrong. It is evident by so many recent attacks since mid 1980's that bioterrorism is not a myth but a real practical problem. Biotechnology can be used by committed terrorist groups quite easily to produce microorganisms that are capable of large scale morbidity and mortality.
The five basic attributes that characterize a perfect military biological warfare (BW) agent are as follows:
a) High virulence coupled with high host specificity;
b) High degree of controllability; the organism should attack only specific groups or populations of people and should not attack the people initiating the bioterrorist attack.
c) High degree of resistance to adverse environmental forces;
d) Lack of timely countermeasures to the attacked population;
e) Ability to camouflage the BW agent with relative ease.
Some of these attributes might not be so important for BW agents that will be applied for terrorist purposes. For example, a terrorist group might be unconcerned whether or not the agents it uses can be controlled after release. Nevertheless, these criteria serve as useful considerations regarding the type of microorganisms which can possibly be used by bioterrorists. In addition, to develop perfect bioterrorist agents, modern biotechnology techniques may be applied to enhance any or all of eight characteristics or traits of microorganisms i.e.- hardiness, resistance, infectiousness, pathogenicity, specificity, detection avoidance, senescence, and the viable but non-culturable state (5).
Use of molecular biology in enhancing bioterrorist weapons
In 2001, Australian scientists manipulated the mousepox virus to suppress the wild mouse population. The outcome was a modified virus that was far deadlier than the original one. This modified strain was also capable of killing mice naturally immune to mousepox or those immunized against the mousepox virus. Since the smallpox and the mousepox viruses are analogous to each other, it is entirely possible that the same experiment can be carried out in the smallpox virus. The smallpox virus is not readily available to terrorist organizations, however it is possible for them to modify other viruses to subvert the human immune system. Again, it is not impossible to synthesize a new organism. In 2002, scientists in USA were successful in synthesizing polio virus from scratch using chemicals available in the open market.
It is not only viruses that are prone to genetic manipulation. Bacteria and Mycobacteria are also prone to genetic modification. Mycobacteria have been manipulated and a hypervirulent mutant strain of tuberculosis has been produced. Similar experiments have been carried out with protozoa like Leishmania major. It is therefore possible to create lethal microorganisms using easily available methods. It would be wrong to assume that the methods would be limited to research laboratories. Most of the techniques used are easily available and can be reproduced in the average laboratory.
The molecular basis of detection
It is easy for the bioterrorist to manipulate the microscopic world for his benefits. However, it is equally easy for the biotechnologist to detect the organism and institute appropriate actions. Ideally, detection platforms should be capable of rapidly detecting and confirming biothreat agents, including modified or previously uncharacterized agents, directly from complex matrix samples, with no false results. Furthermore, the instrument should be portable, user-friendly, and capable of testing for multiple agents simultaneously. Such an instrument is yet unavailable.
The PCR can be used for the detection of bioterrorist weapons. However, it has its own inherent problems which have been alluded to earlier. It also requires a clean sample and is unable to detect protein toxins. It can also give a false positive result because of its inherent sensitivity. Q-PCR can be utilized to detect several targets simultaneously using different reporter dyes for different targets. However, accurate characterization or identification of bacteria by Q-PCR is limited by the same bias and variations that are inherent in many nucleic acid techniques.
Immunoassays have increasingly been used and developed for detection of infectious diseases. Immunological detection has been successfully employed for detection of biothreat agents such as bacterial cells, spores, viruses, and toxins based on the concept that any compound capable of triggering an immune response can be targeted as an antigen. Immunoassays generally test for only one analyte per assay. The specificity of immunoassays is limited by antibody quality, and sensitivity. The sensitivity is typically lower than with PCR and other DNA-based assays. As improvements are made in antibody quality (e.g., production of antibodies from recombinant libraries) and in assay parameters, it may be possible to increase immunoassay sensitivity and specificity. It is also proposed to use antibody fragments for detection.
Aptamers are small DNA or RNA ligands that recognize a target by shape and not by sequence. RNA aptamers includes the ribozymes that can be engineered to generate a signal after target capture. DNA aptamers bind to a target after exposure to UV light. Aptamers can be used to detect entire organisms like Bacillus anthracis spores or toxins like ricin.
Finally, the microarray. Microarrays can potentially detect several organisms at one go, therefore it remains potentially the tool of the future in detecting biological warfare agents.
To conclude, It is possible for committed terrorists to manipulate microorganisms using available molecular techniques to make them more virulent without much difficulty. However, alertness and access to modern diagnostic methods can easily halt a bioterrorist attack. In conclusion, it can be stated that the key components to the fight against a bioterrorist attack are preparedness and awareness.